Protein-tyrosine kinases (PTKs) are an important class of therapeutic targets. Virtually all clinically useful kinase inhibitors to date compete for ATP binding at the kinase domain active site. However, the high degree of kinase sequence and structural homology limits the selectivity of ATP-competitive kinase inhibitors. In this proposal, we seek to develop a completely new screening approach for the discovery of selective inhibitors for protein-tyrosine kinases of the non-receptor class. We aim to discover small molecules that enhance the natural mechanisms associated with kinase domain regulation. Allosteric inhibitors that target unique structural features outside of the kinase domain are likel to provide greater specificity for their intended kinase targets, and may also stabilize kinase domain conformations that promote the action of existing inhibitors. We will validate these concepts using conformationally restricted forms of the oncogenic protein-tyrosine kinase Bcr-Abl as a test case, and in parallel develop novel screening strategies to identify small molecule allosteric sensitizers/inhibitors for this oncoprotein. In the first aim, we will test the hypothess that intramolecular interactions can allosterically enhance the actions of conformationally selective inhibitors. The Bcr-Abl kinase core consists of regulatory SH3 and SH2 modules, followed by the catalytic kinase domain. By binding to the linker connecting the SH2 and kinase domains, the SH3 domain allosterically represses Abl kinase activity. Bcr fusion destabilizes this interaction, resulting in a constitutively active kinase. Here we will test the effects of engineerd enhancement of SH3:linker interaction on Bcr-Abl biological function and sensitivity to conformationally sensitive inhibitors that target the kinase domain. Results of these studies will support high-throughput screening (HTS) campaigns to identify small molecules that enhance SH3:linker interaction as allosteric inhibitors of Bcr-Abl. The second aim will test the hypothesis that a high throughput screen (HTS) can be developed to identify compounds which enhance natural intramolecular regulatory interactions. In this Aim, we will develop novel recombinant probe proteins based on the Abl SH3-SH2-linker regulatory apparatus (Abl 32L proteins). Our previous work shows that these constructs can be readily expressed in bacteria in a structurally competent state, providing an excellent starting point for novel single- molecule fluorescence polarization and/or FRET HTS assay development. Pilot-scale chemical library screens will be performed with these Abl-32L probe proteins, and 'hit' compounds tested for their effects on Bcr-Abl kinase activity and kinase domain inhibitor sensitivity both in vitro and n cell-based assays. These pilot studies will be leveraged in future large-scale HTS campaigns, structural and mechanistic studies, and through adaptation to other multi-domain kinase systems with therapeutic relevance (e.g., Fes, Src and Tec/Btk kinase families).